To study tissue-mediated control of immunity, we have used 4 different systems. 1. IMMUNITY &HEALING: The Danger model led us to the view that tissues influence the effector class of immunity in order to prevent immune-mediated local damage. To determine whether the healing process in wounded tissues is affected by the immune system, we punched small holes in the ears of wild type and mutant mice and measured the healing rate. To our surprise, we found that middle-aged mice quickly regenerate all of the ear tissues (including the cartilage) so well that there is eventually no visible scar. We tested many mutant strains of mice and found that there is essentially no difference in the regenerative capacity of middle aged mice of most strains, regardless of the presence or absence of various cells and/or molecules involved in immunity. Histology suggested that the amount of inflammation is different between the young and middle-aged mice. Thus we tested NSAIDs and found that they greatly inhibit the regeneration, while croton oil enhances it. Thus, contrary to common opinion, it appears that inflammation enhances regeneration. Using Doppler and MRI, we found that the extent of swelling is greater in middle-aged mice. As swelling occuring during embryogenesis causes cells to release from the inhibition of contact with the extracellular matrix, we suggest that swelling may have the same effect in adult ears: the more swelling, the greater the release from contact inhibition, and the greater the potential for regeneration. 2. DENDRITIC CELLS &IL-12p70: It is currently thought that dendritic cells become exhausted within 24 hours of stimulation by LPS. We have found that this is incorrect. Although 24hr-stimulated dendritic cells do not produce p70 upon further restimulation by LPS, they can produce good quantities of IL-12p70 when re-stimulated by activated T cells. We found that TH1 cells and TH0 cells can stimulate this production of IL-12p70 while TH2 cells cannot. We can mimic the activated T cells with combinations of various cytokines in the presence of CD40-L. Surprisingly, the combination of CD40-L and IL-4 induces IL-12p70, though Th2 cells, which have both molecules, do not. We found that the inability of Th2 cells to induce IL-12p70 is due to their concomitant production of IL-10. The 'exhausted'dendritic cells make a host of cytokines and chemokines not made by freshly stimulated dendritic cells. Thus dendritic cells are not in control of immune effector class, but instead relay signals from the cells with which they are in contact. 3. ORAL TOLERANCE TO LATE-APPEARING ANTIGENS: One of the problems with the self-non-self model is that it does not account for tolerance to antigens that appear late in life, such as the milk proteins of lactating mothers (eg. alpha lactalbumin, beta lactoglobulin, casein etc.). To see if tolerance to these proteins is established in the thymus, or perhaps by fetal exposure during pregnancy or neonatal exposure during lactation, we have been using a model system using mice that have had their alpha lactalbumin gene replaced with the human homologue (we call these hALAC mice). To make experimental animals, we bred normal B10.BR females to B10.BR hALAC males. The F1 progeny carry both human and mouse alleles of ALAC but have only been exposed to mALAC from their mothers. When immunized to hALAC after weaning but before puberty, these mice (both males and females) make good T cell and antibody responses. However, when these F1 mice, or normal mice are transferred just after birth to hALAC mothers, they cannot be immunized until some time after weaning. Thus, tolerance is caused by the oral ingestion of hALAC during suckling. We are now following these data to see how long the oral tolerance effect lasts. 4. THE GUT: a. EFFECT OF COMMENSAL BACTERIA ON THE HEALTH OF INTESTINAL TISSUE: we are studying, by microarray analysis of laser microdissected sections of small intestine, the response of various tissues (eg lamina propria, epithelium, crypts) to the presence or absence of commensal bacteria. Preliminary data suggest that 1) many more cells produce anti-microbial peptides than previously thought. 2) antimicrobial peptides are made by epithelial cells, not just Paneth cells b. THE EFFECT OF ANTIBIOTICS ON THE HEALTH OF INTESTINAL TISSUE. Mice were given a cocktail of antibiotics for several weeks and then jejunum tissue was assessed by microarray analysis. We found that antibiotics cause expression changes in a number of genes expressed by normal gut epithelium. Some of these are indirect effects due to the loss of commensal bacteria. Others, however, are direct effects on jejunal tissue, as the changes were also found in antibiotic-treated germ free mice. These direct effects were mostly concentrated in mitochondrial functions. c. THE EFFECT OF B CELLS ON THE HOMEOSTASIS OF THE GUT. Preliminary results suggest that in the absence of B cells, fat metabolism and leptin levels are affected. Thus B cells have a strong effect on normal gut function. We are now analyzing the gut flora in B cell containing and mutant mice to see if the effect of B cells is direct or indirect via a change in the flora. 5. A SPONTANEOUS MUTANT B CELL DEFICIENT MOUSE: In the process of analyzing the effect of B cells on the gut, we discovered a spontaneous mutant mouse deficient in B cells. We characterized these mice and found that the defect was in the Cd79b gene, and that this led to a block in B-cell development at the early C'stage.